CO-CLUSTERING OF BETA-1-INTEGRINS, CYTOSKELETAL PROTEINS, AND TYROSINE-PHOSPHORYLATED SUBSTRATES DURING INTEGRIN-MEDIATED LEUKOCYTE AGGREGATION

The involvement of the VLA-4 integrin in an alternative leukocyte homo typic adhesion mechanism that is LFA-1/ICAM-1 independent, has been pr eviously reported. We describe here the localization of beta1 and alph a4 integrin subunits at sites of cell-cell contact, on both beta1- and alpha4-induced aggregates of B lymphoblastoid Ramos cells. Moreover, the distribution of different VLA-alpha subunits was also examined on beta1-induced cell aggregates of alpha2- and alpha4-transfected K-562 cells. Both alpha2 and alpha4 integrin subunits were mainly localized at sites of intercellular boundaries, suggesting a possible role for t hese integrins in leukocyte intercellular adhesion. The fibronectin re ceptor alpha5 subunit was either diffuse throughout the plasma membran e, or displayed some accumulation at sites of cell-cell contact. Even though homotypic aggregation of U-937 cells was induced with the anti- alpha5 P1 D6 mAb, the alpha5 subunit showed only partial redistributio n to regions of cell-cell contact, compared with the complete redistri bution of the alpha4 subunit in the alpha4-induced aggregates. The reo rganization of the actin-cytoskeleton was observed at sites of interce llular boundaries in both the anti-beta1- and anti-alpha4-induced cell aggregates. Hence, F-actin and the cytoskeletal protein talin co-loca lized with beta1 and alpha4 integrin clusters at sites of cell-cell co ntact. Signal transduction during VLA-mediated homotypic cell adhesion has also been investigated. We found co-localization of beta1 and alp ha4 subunits with tyrosine-phosphorylated proteins at cell-cell contac t regions during cell aggregation. These data indicate that VLA integr in-mediated leukocyte aggregation results in clustering of beta1-integ rins at sites of cell-cell contact, together with co-localization of c ytoskeletal proteins. These results also suggest that protein tyrosine phosphorylation is an important signal transduction mechanism when VL A integrins participate in intercellular contacts.